The invention relates to proteins having A and B chains of a ricin-like toxin, linked by a linker sequence which is specifically cleavable by a retroviral protease to release the active A chain. The invention also relates to a nucleic acid molecule encoding the protein and to expression vectors incorporating the nucleic acid molecule. Also provided is a method of inhibiting or destroying mammalian cells infected with a retrovirus utilizing the proteins of the invention and pharmaceutical compositions for treating HIV infection.
Bacteria and plants are known to produce cytotoxic proteins which may consist of one, two or several polypeptides or subunits. Those proteins having a single subunit may be loosely classified as Type I proteins. Many of the cytotoxins which have evolved two subunit structures are referred to as type II proteins(Saelinger, C. B. in Trafficking of Bacterial Toxins (eds. Saelinger, C. B.) 1-13 (CRC Press Inc., Boca Raton, Fla., 1990). One subunit, the A chain, possesses the toxic activity whereas the second subunit, the B chain, binds cell surfaces and mediates entry of the toxin into a target cell. A subset of these toxins kill target cells by inhibiting protein biosynthesis. For example, bacterial toxins such as diphtheria toxin or Pseudomonas exotoxin inhibit protein synthesis by inactivating elongation factor 2. Plant toxins such as ricin work by directly inactivating ribosomes [Olsnes, S. and Phil, A. in Molecular action of toxins and viruses (eds. Cohen, P. and vanHeyningen, S.); 51-105 (Elsevier Biomedical Press, Amsterdam, 1982].
Ricin, derived from the seeds of Ricinus communis (castor oil plant), is the most potent of the plant toxins. It is estimated that a single ricin A chain is able to inactivate ribosomes at a rate of 1500 ribosomes/minute. Consequently, a single molecule of ricin is enough to kill a cell (Olsnes, S. and Phil, A. in Molecular action of toxins and viruses (eds. Cohen, P. and vanHeyningen, S.) 51-105 (Elsevier Biomedical Press, Amsterdam, 1982). The ricin toxin is a glycosylated heterodimer with A and B chain molecular masses of 30,625 Da and 31,431 Da respectively. The A chain of ricin has an N-glycosidase activity and catalyzes the excision of a specific adenine residue from the 28S rRNA of eukaryotic ribosomes (Endo, Y; and Tsurugi, K. J. Biol. Chem. 262:8128 (1987)). The B chain of ricin, although not toxic in itself, promotes the toxicity of the A chain by binding to galactose residues on the surface of eukaryotic cells and stimulating receptor-mediated endocytosis of the toxin molecule (Simmons et al. Biol. Chem. 261:7912 (1986)).
Protein toxins are initially produced in an inactive, precursor form. Ricin is initially produced as a single polypeptide (preproricin) with a 35 amino acid N-terminal presequence and 12 amino acid linker between the A and B chains. The pre-sequence is removed during translocation of the ricin precursor into the endoplasmic reticulum (Lord, J. M. Eur. J. Biochem. 146:403-409 (1985) and Lord, J. M. Eur. J. Biochem. 146:411-416 (1985)). The proricin is then translocated into specialized organelles called protein bodies where a plant protease cleaves the protein at a linker region between the A and B chains (Lord, J. M. et al., FASAB Journal 8:201-208 (1994)). The two chains, however, remain covalently attached by an interchain disulfide bond (cysteine 259 in the A chain to cysteine 4 in the B chain) and mature disulfide linked ricin is secreted from the plant cells. The A chain is inactive in the proricin (O""Hare, M., et al. FEBS Lett. 273:200-204 (1990)) and it is inactive in the disulfide-linked mature ricin(Richardson, P. T., et al. FEBS Lett. 255:15-20 (1989)). The ribosomes of the castor bean plant are themselves susceptible to inactivation by ricin A chain; however, as there is no cell surface galactose to permit B chain recognition the A chain cannot re-enter the cell. The exact mechanism of A chain release and activation in target cell cytoplasm is not known (Lord, J. M. et al., FASAB Journal 8:201-208 (1994)). However, it is known that for activation to take place the disulfide bond between the A and B chains must be reduced and, hence, the linkage between subunits broken.
The ricin gene has been cloned and sequenced, and the X-ray crystal structures of the A and B chains have been described (Rutenber, E., et al. Proteins 10:240-250 (1991); Weston et al., Mol. Bio. 244:410-422, 1994; Lamb and Lord Eur. J. Biochem. 14:265 (1985); Halling, K., et al. Nucleic Acids Res. 13:8019 (1985)). Due to its extreme toxicity there has been much interest in making ricin-based immunotoxins as therapeutic agents for destroying or inhibiting target cells or organisms (Vitetta et al., Science 238:1098-1104(1987)). An immunotoxin is a conjugate of a specific cell-binding component, such as a monoclonal antibody or growth factor and the toxin in which the two protein components are covalently linked. Generally, the components are chemically coupled. However, the linkage may also be a peptide or disulfide bond. The antibody directs the toxin to cell types presenting a specific antigen thereby providing a specificity of action not possible with the natural toxin. Immunotoxins have been made both with the entire ricin molecule (i.e. both chains) and with the ricin A chain alone ( Spooner et al. Mol. Immunol. 31:117-125, (1994)).
Class 2 ribosomal inhibitory proteins (RIP-2) constitute other toxins possessing distinct functional domains for cytotoxicity and cell binding/toxin translocation which include abrin, modeccin, volkensin, (Sandvig, K. et al., Biochem. Soc. Trans. 21:707-711 (1993)) and mistle toe lectin (viscumin) (Olsnes, S. and Phil, A. in Molecular action of toxins and viruses (eds. Cohen, P. and vanHeyningen, S.) 51-105 Elsevier Biomedical Press, Amsterdam, 1982; Fodstad et al. Canc. Res. 44: 862 (1984)).
Immunotoxins made with the ricin dimer (IT-Rs) are more potent toxins than those made with only the A chain (IT-As). The increased toxicity of IT-Rs is thought to be attributed to the dual role of the B chains in binding to the cell surface and in translocating the A chain to the cytosolic compartment of the target cell (Vitetta et al., Science 238:1098-1104(1987); Vitetta and Thorpe Seminars in Cell Biology 2:47-58 (1991)). However, the presence of the B chain in these conjugates also promotes the entry of the immunotoxin into nontarget cells. Even small amounts of B chain may override the specificity of the cell-binding component as the B chain binds nonspecifically to N-glycosylated galactose, present on most cells. IT-As are more specific and safer to use than IT-Rs. However, in the absence of the B chain the A chain has greatly reduced toxicity.
A number of immunotoxins have been designed to recognize antigens on the surfaces of tumour cells. A major problem with the use of ITs is that often the target antigen is also found on non-tumour cells (Vitetta et al., Immunology Today 14:252-259 (1993)). Also, due to the reduced potency of IT-As as compared to ITRs, large doses of IT-As must be administered to patients. The large doses frequently cause immune responses and production of neutralizing antibodies in patients (Vitetta et al., Science 238:1098-1104(1987)). IT-As and IT-Rs both suffer from reduced toxicity as the A chain is not released from the conjugate into the target cell cytoplasm.
The insertion of intramolecular cleavage sites between the cytotoxic and cell-binding components of a toxin can mimic the way that the natural toxin is activated. European patent application no. 466,222 describes the use of maize-derived pro-proteins which can be converted into active form by cleavage with extracellular blood enzymes such as factor Xa, thrombin or collagenase. Westby et al. (Bioconjugate Chem., 3:375-381, 1992) documented fusion proteins which have a specific cell binding component and proricin with a protease sensitive cleavage site specific for factor Xa within the linker sequence. O""Hare et al. (FEBS Lett. 273:200-204, 1990) also describe a recombinant fusion protein of RTA and staphylococcal protein A joined by a trypsin-sensitive cleavage site. In view of the prevalence of the extracellular proteases utilized in these approaches, such artificial activation of the toxin precursor or immunotoxin do not confer a mechanism for intracellular toxin activation, and the problems of target specificity and adverse immunological reactions to the cell-binding component of the immunotoxin remain.
In view of the extreme toxicity of proteins such as ricin, the lack of specificity of the immunotoxins may severely limit their usefulness as therapeutics for the treatment of cancer and infectious diseases. The preparation of a suitable specific cell binding component may be problematic. For example, antigens specific for the target cell may not be available and many potential target cells and infective organisms can alter their antigenic make up rapidly to avoid immune recognition.
The potential of bacterial and plant toxins for inhibiting mammalian retroviruses, particularly AIDS, has been investigated. Bacterial toxins such as Pseudomonas exotoxin-A and subunit A of diphtheria toxin; dual chain ribosomal inhibitory plant toxins, such as ricin and single chain ribosomal inhibitory proteins such as trichosanthin and poke weed antiviral protein have been used for the elimination of HIV infected cells (Olson et al. 1991, AIDS Res. and Human Retroviruses 7:1025-1030). The high toxicity of these toxins for mammalian cells, combined with a lack of specificity of action poses a major problem to the development of pharmaceuticals incorporating the toxins, such as immunotoxins.
Immunotoxins are designed such that their specificity of action is determined solely by the antibody component; antigen presenting cells are preferentially destroyed by the drug (Pastan et al., Annals New York Academy of Sciences 758:345-353 (1995)). The toxin protein of immunotoxin conjugates does not give the therapeutic any additional specificity of action; it will bring about the destruction of any cell it is delivered to.
The present inventors have prepared novel recombinant toxic proteins which are specifically toxic to cells infected with retroviruses and which do not depend for their specificity of action on a specific cell-binding component. The recombinant proteins of the invention have an A chain of a ricin-like toxin linked to a B chain by a linker sequence, which may be specifically cleaved by a retroviral protease within infected cells to activate the toxic A chain.
In one aspect, the present invention provides a purified and isolated nucleic acid having a nucleotide sequence encoding an A chain of a ricin-like toxin, a B chain of a ricin-like toxin and a heterlogous linker amino acid sequence, linking the A and B chains. The linker sequence is not a linker sequence of a ricin-like toxin, but rather the heterologous linker sequence contains a cleavage recognition site for a retroviral protease. The A and or the B chain may be those of ricin.
In an embodiment, the cleavage recognition site is the cleavage recognition site for an HIV protease. In a particular embodiment, the linker amino acid sequence comprises VSQNYPIVQNFN (SEQ.ID.NO: 20); SKARVLAEAMSN (SEQ.ID.NO: 21); or SIRKILFLDGIN (SEQ.ID.NO: 22). In further particular embodiments, the nucleic acid has the nucleotide sequence shown in FIG. 8 (SEQ.ID.NO: 23), FIG. 9 (SEQ.ID.NO: 24) or FIG. 10 (SEQ.ID.NO: 25).
In another embodiment, the cleavage recognition site is the cleavage recognition site for a human T-cell leukemia virus protease. In a particular embodiment, the linker amino acid sequence comprises SAPQVLPVMHPN (SEQ.ID.NO: 55) or SKTKVLVVQPKN (SEQ.ID.NO: 56) cleaved by a human T-cell leukemia virus-I (HTLV-I) protease; or, SKTKVLVVQPRN (SEQ.ID.NO: 57) or STTQCFPILHPN (SEQ.ID.NO: 58) cleaved by a human T cell leukemia virus-II (HTLV-II) protease.
The present invention further provides a plasmid incorporating the nucleic acid of the invention. In an embodiment, the plasmid has the restriction map as shown in FIG. 1A, 2A, 3A, 16A, 17A, 18A, or 19A.
In another embodiment, the present invention provides a baculovirus transfer vector incorporating the nucleic acid of the invention. In particular embodiments, the invention provides a baculovirus transfer vector having the restriction map as shown in FIGS. 5, 6, 7, 16C, 17C, 18C, or 19C or having the DNA sequence as shown in FIG. 11 (SEQ.ID.NO: 26).
In a further aspect, the present invention provides a recombinant protein comprising an A chain of a ricin-like toxin, a B chain of a ricin-like toxin and a heterologous linker amino acid sequence, linking the A and B chains, wherein the linker sequence contains a cleavage recognition site for a retroviral protease. The A and or the B chain may be those of ricin.
In another aspect, the invention provides a method of inhibiting or destroying mammalian cells infected with a retrovirus having a protease, comprising the steps of preparing a recombinant protein of the invention having a heterologous linker sequence which contains a cleavage recognition site for the retrovirus protease and introducing the recombinant protein into the cells. In an embodiment, the retrovirus is HIV.
The present invention also relates to a method of treating a mammal infected with HIV by administering the recombinant proteins of the invention to the mammal.
Also provided is a process for preparing a pharmaceutical for treating a mammal infected with a retrovirus having a protease comprising the steps of preparing a purified and isolated nucleic acid having a nucleotide sequence encoding an A chain of a ricin-like toxin, a B chain of a ricin-like toxin and a heterologous linker amino acid sequence, linking the A and B chains, wherein the linker sequence contains a cleavage recognition site for the protease; introducing the nucleic acid into a host cell; expressing the nucleic acid in the host cell to obtain a recombinant protein comprising an A chain of a ricin-like toxin, a B chain of a ricin-like toxin and a heterlogous linker amino acid sequence, linking the A and B chains wherein the linker sequence contains the cleavage recognition site for the protease; and suspending the protein in a pharmaceutically acceptable carrier, diluent or excipient.
In an embodiment, a process is provided for preparing a pharmaceutical for treating a mammal infected with a retrovirus having a protease comprising the steps of identifying a cleavage recognition site for the protease; preparing a recombinant protein comprising an A chain of a ricin-like toxin, a B chain of a ricin-like toxin and a heterlogous linker amino acid sequence, linking the A and B chains wherein the linker sequence contains the cleavage recognition site for the protease and suspending the protein in a pharmaceutically acceptable carrier, diluent or excipient.
In a further aspect, the invention provides a pharmaceutical composition for treating a retroviral infection, such as HIV, in a mammal comprising the recombinant protein of the invention and a pharmaceutically acceptable carrier, diluent or excipient.
The invention also contemplates a method for treating cancer cells containing an HTLV protease comprising (a) preparing a recombinant protein of the invention having a heterologous linker sequence which contains a cleavage recognition site for an HTLV protease; an (b) introducing the recombinant protein into the cells. The method can be used to treat a mammal with human T-cell leukemias involving HTLV. Compositions for treating human T-cell leukemias involving HTLV comprising the recombinant protein of the invention having a heterologous linker sequence which contains a cleavage recognition site for an HTLV protease, and a pharmaceutically acceptable carrier, diluent, or excipient are also provided.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.